The long term objective of the work is to understand at the molecular level how tumor promoters in particular the phorbol ester 12otetradecanoylphorbol13acetate (TPA), interact with cell and virus genes to produce a transformed phenotype in cells exposed to an initiating carcinogenic stimulus. This proposal continues to test the general hypothesis that TPA stimulates the transcription of specific genes by altering the activity and/or specificity of RNA polymerase II, and to examine the mechanism of this effect. The proposed experiments will attempt to identify components of the regulatory pathway as well as targets associated directly with the process of transcription. Successful conclusion of the experiments will provide an in vitro test for the regulation of cloned cell sequences responsive to TPA in vivo, and will lay the groundwork for complete in vitro reconstruction of a biologically important transmembrane signaling pathway. The proposed experiments will characterize the requirements for stimulation of in vitro transcription of the type 5 human adenovirus (Ad5) immediate early gene (E1A) by activated endogenous protein kinase C in whole cell extracts and by purified Ckinase in reconstructed reactions using purified RNA polymerase II plus cell fractions containing partially purified transcription factors. Experiments will attempt to identify a phosphoprotein target for Ckinase in in crude extracts; in purified fractions that stimulate transcription activity; on isolate transcription complexes from Ad5infected cells and from in vitro transcription reactions; and from the chromain of TPAtreated cells. Phosphoproteins from labeled transcription complexes will be characterized by two dimensional polyacrylamide gel electrophoresis and autoradiography. The effect of Ckinase on in vitro transcription of other TPAinducible genes will be tested.